Polymerization of the protein actin into helical filaments powers the directed motility of eukaryotic cells and some pathogenic bacteria. Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. The essential regulatory protein, cofilin, is one of four actin-binding proteins that precisely choreograph actin assembly and organization in living systems. It acts by severing filaments, which increases the concentration of filament ends available for subunit addition and dissociation, thereby accelerating overall actin network dynamics and reorganization. It is therefore of general medical importance to understand how cofilin fragments actin filaments. Although the effects of cofilin binding to actin filaments have been extensively studied, the molecular mechanism of how cofilin severs filaments, which have stiffness comparable to commercial laboratory plastics, remains a central and unresolved mystery of cellular actin cytoskeleton reorganization. Elucidating the cofilin severing mechanism demands a multi-disciplinary approach integrating biology, chemistry, physics and mathematical modeling. Proposed research efforts focus on identifying how specific cation binding, post-translational modification, competition with other regulatory proteins, and filament shape deformations modulate actin filament structure and severing by vertebrate cofilin. Five general hypotheses will be tested. The first is that vertebrate cofilin severs filaments by dissociating a specific filamen-associated cation that controls filament structure and mechanical properties. The second is that competitive displacement of cofilin by other filament binding proteins can promote cofilactin filament severing by introducing boundaries of bare and cofilin-decorated segments. The third is that phosphorylation enhances cooperative cofilin binding and inhibits severing, not by lowering cofilin occupancy along filaments, but by reducing the density of boundaries where severing can occur. The fourth is that contractile protein- driven deformations in filament shape enhance severing by cofilin. The fifth is that actin filaments can act as tension sensors that recruit or exclude cofilin depending on the magnitude and mode of filament shape deformation. We will integrate biochemical and biophysical approaches, including experimental manipulation of single filaments, with mathematical modeling and simulations to develop predictive molecular models of actin filament elasticity and fragmentation, and directly test hypotheses formulated from biochemical and biophysical analysis of cofilin-actin interactions completed during the prior award period. The proposed research activities will advance knowledge of actin filament physiology by providing multi-scale relationships between filament mechanics, structure, and the biological function (e.g. severing activity) of essential regulatory proteins. New experimental and methods of analysis readily applicable to other filament binding proteins will be developed. Novel insight regarding the relationship between actin filament elasticity, conformation and regulatory protein occupancy will emerge from the work.